Microfluidic single cell sorting system and sorting method based on acoustic wave focusing and sorting

Abstract

The present invention discloses a microfluidic single cell sorting system and a sorting method based on acoustic wave focusing and sorting, wherein the system comprises a microfluidic chip, an acoustic wave sorting module, and a fluorescence detection module; the microfluidic chip comprises a substrate, a hydraulic focusing structure for cell focusing, an acoustic wave focusing flow channel, and a sorting flow channel, wherein the hydraulic focusing structure, the acoustic wave focusing flow channel, and the sorting flow channel are sequentially connected. The present invention adopts an LED light source as an excitation light source, and directly uses an optical fiber to connect the excitation light source, and the optical fiber is connected to a photon counting photomultiplier tube as a detector for fluorescence signal detection, thereby realizing low data volume collection of fluorescence signals. Based on acoustic wave sorting, cells can be deflected under the action of acoustic wave radiation force, and the number of deflected cells can be accurately controlled to ensure cell activity. Based on an inverted fluorescence microscope, there is no need to build an additional fluorescence detection optical path.

Description

Technical Field

[0001] The invention belongs to the field of microfluidic flow cytometry, and in particular relates to a microfluidic single cell sorting system and a sorting method based on acoustic wave focusing and sorting. Background Art

[0002] Microfluidic flow cytometry is an important technology for analyzing the characteristics and behaviors of single cells and is widely used in biomedical research, clinical diagnosis, and drug development. Existing flow cytometry systems usually rely on hydraulically driven microfluidic chips combined with optical detection devices for high-throughput cell analysis and sorting. However, such systems have certain limitations. Traditional microfluidic flow sorting systems mainly use hydraulic focusing to control the flow of cells, but this method is easily interfered by background noise and cell volume changes during signal acquisition, resulting in low sorting efficiency. In addition, many existing systems use complex and expensive optical detection elements, such as lasers and high-precision photodetectors, which significantly increase the system cost and are not conducive to widespread application.

[0003] In recent developments, acoustic focusing technology has been gradually applied to the field of microfluidics. Compared with traditional hydraulic focusing, acoustic focusing has the advantages of non-contact, low energy consumption, and high precision. However, existing systems based on acoustic focusing still have some problems, such as complex design, weak detection signal, and low signal-to-noise ratio.

[0004] Therefore, the current microfluidic flow cytometry system still has room for improvement in terms of detection signal quality, cost control, and integration. In order to overcome these deficiencies in the prior art, the present invention proposes a microfluidic single cell sorting system and a sorting method based on acoustic wave focusing and sorting, aiming to improve the signal-to-noise ratio, reduce system costs, and achieve higher integration. Summary of the invention

[0005] In response to the above problems, the present invention proposes a microfluidic single-cell sorting system and a sorting method based on acoustic wave focusing and sorting, which integrates hydraulic focusing and acoustic wave focusing, does not require an oil phase, and has the characteristics of high sorting accuracy and low system complexity.

[0006] The technical solution adopted by the present invention to solve its technical problem is: In one aspect, the present invention provides a microfluidic single cell sorting system based on acoustic wave focusing and sorting, the system comprising a microfluidic chip, an acoustic wave sorting module, and a fluorescence detection module; The microfluidic chip comprises a substrate, a hydraulic focusing structure for cell focusing, an acoustic wave focusing channel, and a sorting channel, wherein the hydraulic focusing structure, the acoustic wave focusing channel, and the sorting channel are sequentially connected, and the axis of the hydraulic focusing structure coincides with the acoustic wave focusing channel; The acoustic wave sorting module includes two acoustic wave focusing electrodes and an acoustic wave sorting electrode for cell sorting. The two acoustic wave focusing electrodes are connected to both sides of the acoustic wave focusing flow channel on the substrate near the hydraulic focusing structure. The acoustic wave sorting electrode is connected to one side of the acoustic wave focusing flow channel on the substrate and the intersection of the acoustic wave focusing flow channel and the sorting flow channel. The number of the acoustic wave sorting electrodes is the same as the number of the sorting flow channels. The fluorescence detection module includes a laser optical fiber connected to an LED light source, a detection optical fiber connected to a photomultiplier tube, a data acquisition card for real-time recording the number of level signals output by the photomultiplier tube, a host computer for reading data from the data acquisition card, and an inverted fluorescence microscope for fixedly installing a microfluidic chip. The laser optical fiber and the detection optical fiber are located on one side of an acoustic wave focusing channel and between an acoustic wave focusing electrode and an acoustic wave sorting electrode.

[0007] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the acoustic wave sorting module also includes a signal generator for outputting electrical signals, a power amplifier for amplifying the output signal of the signal generator, and a DC power supply for providing an operating voltage for the power amplifier.

[0008] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the hydraulic focusing structure includes a sheath liquid inlet, a sample liquid inlet, a hydraulic focusing point, a sheath liquid delivery channel, and a hydraulic focusing flow channel. The sheath liquid inlet is connected to the sheath liquid delivery channel, and the sheath liquid delivery channel is transformed from one inlet to a first sheath liquid delivery channel and a second sheath liquid delivery channel that are symmetrical about the hydraulic focusing flow channel. The sample liquid inlet is connected to the hydraulic focusing flow channel, and the ends of the first sheath liquid delivery channel, the second sheath liquid delivery channel, and the hydraulic focusing flow channel intersect and penetrate to form a hydraulic focusing point. The hydraulic focusing point is connected to the acoustic wave focusing flow channel, and the ends of the acoustic wave focusing flow channel are respectively connected to the waste liquid outlet and the sorting flow channel.

[0009] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, there are two acoustic wave sorting electrodes, which are respectively located on both sides of the acoustic wave focusing channel. The two sides of the acoustic wave focusing channel near the end are connected to the first sorting outlet and the second sorting outlet respectively through the first sorting channel and the second sorting channel with the same width for collecting the target liquid, and the waste liquid outlet is connected in the middle near the end.

[0010] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, a pressure relief port is provided between the acoustic wave focusing electrode and the laser optical fiber on the acoustic wave focusing channel, and the pressure relief port is located on the side of the laser optical fiber and the detection optical fiber close to the hydraulic focusing structure.

[0011] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the sheath fluid inlet and the sample fluid inlet are both provided with a filtering microcolumn array.

[0012] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the filtering microcolumn array includes a plurality of filtering microcolumns made of PDMS, and the cross-section of the filtering microcolumns is square, diamond or circular.

[0013] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the acoustic wave focusing electrode and the acoustic wave sorting electrode are interdigitated electrodes.

[0014] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the substrate is assembled by chemically bonding a lithium niobate substrate and polydimethylsiloxane.

[0015] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the bonding method is specifically oxygen plasma bonding.

[0016] On the other hand, the present invention provides a microfluidic single cell sorting method based on acoustic wave focusing and sorting, using the above-mentioned sorting system, comprising the following steps: Step S1, the microfluidic chip is mounted on an inverted fluorescence microscope; Step S2, turning on the power supply and setting the parameters of each electronic instrument; Step S3, the photomultiplier tube receives the fluorescence signal and outputs a corresponding level signal; Step S4, the data acquisition card records the number of levels output by the photomultiplier tube; Step S5, the host computer calculates the photon increment and identifies the fluorescence signal; Step S6, the host computer outputs the sound wave sorting command to the data acquisition card; Step S7, the signal generator receives an external trigger command and outputs an electrical signal; Step S8, the power amplifier amplifies the electrical signal; Step S9: The corresponding cells are sorted to the target outlet by the acoustic radiation force.

[0017] Furthermore, the above-mentioned microfluidic single cell sorting method based on acoustic wave focusing and sorting, The step S2 is to turn on the power supply, set the signal pulse waveform of the signal generator, including the pulse frequency, signal duration and delay trigger time; and adjust the injection speed of the sheath liquid and the sample liquid; In step S4, the counting port of the data acquisition card can continuously record the accumulated values ​​of the TTL level signal output by the photomultiplier tube for collection and processing by the host computer; In step S5, the host computer collects the accumulated number of photons at the counting port of the data acquisition card at a preset sampling time interval, subtracts the accumulated value at the previous moment, and obtains the accumulated number of photons at the sampling time interval; finally, the fluorescence recognition algorithm is used to identify whether there are stained cells flowing through the fluorescence detection area; In step S6, if the host computer detects stained cells, it immediately outputs a sorting command to the data acquisition card; In step S7, after the signal generator receives the sorting pulse signal output by the data acquisition card, it immediately outputs a preset electrical signal to the power amplifier; In step S8, the power amplifier amplifies the electrical signal output by the signal generator, and the amplified signal directly acts on the acoustic wave sorting electrode of the microfluidic chip; In the step S9, when the stained cells flow through the acoustic wave sorting electrode, they are sorted to the corresponding target outlet under the action of the acoustic wave radiation force.

[0018] Furthermore, in the above-mentioned microfluidic single-cell sorting method based on acoustic wave focusing and sorting, in the step S2, the pulse frequency of the signal generator connected to the acoustic wave focusing electrode is 19.7 MHz-19.9 MHz, the pulse frequency of the signal generator connected to the acoustic wave sorting electrode is 39.7 MHz-39.9 MHz, the signal duration is 0.1 ms-10 ms, and the delayed trigger time of the acoustic wave sorting electrode is 1 ms; the injection speed of the sheath liquid is 1 μL / min-3 μL / min, and the injection speed of the sample liquid is 1 μL / min-3 μL / min.

[0019] The beneficial effects of the technical solution of the present invention include at least: The present invention is different from other microfluidic chip flow cytometry detection systems. It uses a low-cost, small-volume LED light source as an excitation light source, and directly uses an optical fiber to connect the excitation light source, and the optical fiber is connected to a photon counting photomultiplier tube as a detector for fluorescence signal detection, so as to achieve low data volume collection of fluorescence signals and reduce the performance requirements of data acquisition devices. At the same time, the fluorescence acquisition system has a simple structure and does not require additional optical components and light path construction.

[0020] The microfluidic chip designed by the present invention is different from other microfluidic flow sorting chips. It uses a combination of hydraulic focusing and acoustic focusing to focus cells, which can effectively improve the detection rate of cells. At the same time, the system only uses one sheath fluid and one sample fluid, which simplifies the operation process and directly detects and analyzes single cells. Different from the traditional oil-in-water droplet generation structure, the present invention achieves efficient cell sorting through acoustic wave focusing and sorting. It does not require oil phase to encapsulate the droplets, can still maintain high cell activity, and avoids pollution from the external environment. Acoustic wave sorting further ensures the activity of cells during the sorting process. This design not only improves the detection accuracy, but also simplifies the assembly and operation of the system, making it easy to promote and apply.

[0021] The acoustic wave sorting proposed in the present invention can make cells deflect under the action of acoustic wave radiation force and can accurately control the number of deflected cells to ensure cell activity.

[0022] The technical solution of the present invention is designed and constructed based on an inverted fluorescence microscope, and the excitation light source and the photomultiplier tube are directly connected with optical fiber. There is no need to build an additional fluorescence detection optical path, which greatly reduces the difficulty and cost of system construction and facilitates the wide application of the system.

[0023] The present invention can be widely used in occasions such as quantitative analysis, concentration analysis, sorting and enrichment of various stained single cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 A schematic diagram of the structure of the sorting system provided by the technical solution of the present invention; Figure 2 A flow chart of the sorting method provided by the technical solution of the present invention; In all the drawings, the same figure marks are used to represent the same elements or structures, wherein: 1-sheath liquid inlet, 2-sample liquid inlet, 3-hydraulic focusing channel, 4-acoustic focusing electrode, 5-acoustic focusing channel, 6-pressure relief port, 7-laser optical fiber, 8-detection optical fiber, 9-acoustic sorting electrode, 10-first sorting outlet, 11-second sorting outlet, 12-waste liquid outlet, 13-first sheath liquid delivery channel, 14-second sheath liquid delivery channel, 15-first sorting channel, 16-second sorting channel, 17-hydraulic focusing point. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0026] In one aspect, the present invention provides a microfluidic single cell sorting system based on acoustic wave focusing and sorting, the system comprising a microfluidic chip, an acoustic wave sorting module, and a fluorescence detection module; The microfluidic chip comprises a substrate, a hydraulic focusing structure for cell focusing, an acoustic wave focusing channel 5, and a sorting channel, wherein the hydraulic focusing structure, the acoustic wave focusing channel 5, and the sorting channel are sequentially connected, and the axis of the hydraulic focusing structure coincides with the acoustic wave focusing channel 5; The acoustic wave sorting module includes two acoustic wave focusing electrodes 4 and an acoustic wave sorting electrode 9 for cell sorting. The two acoustic wave focusing electrodes 4 are connected to both sides of the acoustic wave focusing flow channel 5 on the substrate near the hydraulic focusing structure. The acoustic wave sorting electrode 9 is connected to one side of the acoustic wave focusing flow channel 5 on the substrate and the intersection of the acoustic wave focusing flow channel 5 and the sorting flow channel. The number of the acoustic wave sorting electrodes 9 is the same as the number of the sorting flow channels. The fluorescence detection module includes a laser fiber 7 connected to an LED light source, a detection fiber 8 connected to a photomultiplier tube, a data acquisition card for real-time recording the number of level signals output by the photomultiplier tube, a host computer for reading data from the data acquisition card, and an inverted fluorescence microscope for fixedly mounting the microfluidic chip. The laser fiber 7 and the detection fiber 8 are located on one side of the acoustic wave focusing channel 5 and between the acoustic wave focusing electrode 4 and the acoustic wave sorting electrode 9.

[0027] It should be noted that the sorting channels correspond to the sorting outlets one by one, and the numbers of the acoustic wave sorting electrodes and the sorting channels are the same.

[0028] The laser optical fiber 7 and the detection optical fiber 8 can be located on the same side of the acoustic wave focusing channel 5, or can be located on two separate sides.

[0029] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the acoustic wave sorting module also includes a signal generator for outputting electrical signals, a power amplifier for amplifying the output signal of the signal generator, and a DC power supply for providing an operating voltage for the power amplifier.

[0030] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the hydraulic focusing structure includes a sheath liquid inlet 1, a sample liquid inlet 2, a hydraulic focusing point 17, a sheath liquid delivery channel, and a hydraulic focusing channel 3. The sheath liquid inlet 1 is connected to the sheath liquid delivery channel, and the sheath liquid delivery channel is transformed from one inlet to a first sheath liquid delivery channel 13 and a second sheath liquid delivery channel 14 that are symmetrical about the hydraulic focusing channel. The sample liquid inlet 2 is connected to the hydraulic focusing channel 3. The ends of the first sheath liquid delivery channel 13, the second sheath liquid delivery channel 14, and the hydraulic focusing channel 3 intersect and penetrate to form a hydraulic focusing point 17. The hydraulic focusing point 17 is connected to the acoustic wave focusing channel 5, and the ends of the acoustic wave focusing channel 5 are respectively connected to the waste liquid outlet 12 and the sorting channel.

[0031] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, there are two acoustic wave sorting electrodes 9, which are respectively located on both sides of the acoustic wave focusing channel 5. The acoustic wave focusing channel 5 is connected to the first sorting outlet 10 and the second sorting outlet 11 respectively near the end through the first sorting channel 15 and the second sorting channel 16 of the same width for collecting the target liquid, and the waste liquid outlet 12 is connected in the middle near the end.

[0032] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, a pressure relief port 6 is provided between the acoustic wave focusing electrode 4 and the laser optical fiber 7 on the acoustic wave focusing channel 5, and the pressure relief port 6 is located on the side of the laser optical fiber and the detection optical fiber close to the hydraulic focusing structure.

[0033] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the sheath liquid inlet 1 and the sample liquid inlet 2 are both provided with a filtering microcolumn array.

[0034] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the filtering microcolumn array includes a plurality of filtering microcolumns made of PDMS, and the cross-section of the filtering microcolumns is square, diamond or circular.

[0035] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the acoustic wave focusing electrode 4 and the acoustic wave sorting electrode 9 are interdigitated electrodes.

[0036] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the substrate is assembled by chemically bonding a lithium niobate substrate and polydimethylsiloxane.

[0037] Furthermore, in the above-mentioned microfluidic single-cell sorting system based on acoustic wave focusing and sorting, the bonding method is specifically oxygen plasma bonding.

[0038] On the other hand, the present invention provides a microfluidic single cell sorting method based on acoustic wave focusing and sorting, using the above-mentioned sorting system, comprising the following steps: Step S1, the microfluidic chip is mounted on an inverted fluorescence microscope; Step S2, turning on the power supply and setting the parameters of each electronic instrument; Step S3, the photomultiplier tube receives the fluorescence signal and outputs a corresponding level signal; Step S4, the data acquisition card records the number of levels output by the photomultiplier tube; Step S5, the host computer calculates the photon increment and identifies the fluorescence signal; Step S6, the host computer outputs the sound wave sorting command to the data acquisition card; Step S7, the signal generator receives an external trigger command and outputs an electrical signal; Step S8, the power amplifier amplifies the electrical signal; Step S9: The corresponding cells are sorted to the target outlet by the acoustic radiation force.

[0039] Furthermore, the above-mentioned microfluidic single cell sorting method based on acoustic wave focusing and sorting, The step S2 is to turn on the power supply, set the signal pulse waveform of the signal generator, including the pulse frequency, signal duration and delay trigger time; and adjust the injection speed of the sheath liquid and the sample liquid; In step S4, the counting port of the data acquisition card can continuously record the accumulated values ​​of the TTL level signal output by the photomultiplier tube for collection and processing by the host computer; In step S5, the host computer collects the accumulated number of photons at the counting port of the data acquisition card at a preset sampling time interval, subtracts the accumulated value at the previous moment, and obtains the accumulated number of photons at the sampling time interval; finally, the fluorescence recognition algorithm is used to identify whether there are stained cells flowing through the fluorescence detection area; In step S6, if the host computer detects stained cells, it immediately outputs a sorting command to the data acquisition card; In step S7, after the signal generator receives the sorting pulse signal output by the data acquisition card, it immediately outputs a preset electrical signal to the power amplifier; In step S8, the power amplifier amplifies the electrical signal output by the signal generator, and the amplified signal directly acts on the acoustic wave sorting electrode 9 of the microfluidic chip; In the step S9, when the stained cells flow through the acoustic wave sorting electrode 9, they are sorted to the corresponding target outlet under the effect of the acoustic wave radiation force.

[0040] Furthermore, in the above-mentioned microfluidic single-cell sorting method based on acoustic wave focusing and sorting, in step S2, the pulse frequency of the signal generator connected to the acoustic wave focusing electrode is 19.7 MHz-19.9 MHz, and the pulse frequency of the signal generator connected to the acoustic wave sorting electrode is 39.7 MHz-39.9 MHz, both of which are sinusoidal wave signals; when working, the acoustic wave focusing electrode is always open, the acoustic wave sorting electrode is triggered by the fluorescence signal, the signal duration of the acoustic wave sorting electrode is 0.1 ms-10ms, and the delayed trigger time of the acoustic wave sorting electrode is 1 ms; the injection speed of the sheath fluid is 1 μL / min-3 μL / min, and the injection speed of the sample liquid is 1 μL / min-3 μL / min.

[0041] Specifically, taking two acoustic wave focusing electrodes and two sorting outlets as an example, Figure 1 It is a structural schematic diagram of the sorting system of the present invention. The yellow-filled part in the schematic diagram is the flow channel, which includes: a hydraulic focusing channel 3 and an acoustic focusing channel 5 for realizing cell center focusing. The sheath liquid inlet 1 and the sample liquid inlet 2 are on the same horizontal line, and the initial center focusing of the cell can be achieved by setting the flow rate. The red-filled area 4 is an acoustic focusing electrode 4, and the two acoustic focusing electrodes 4 are symmetrically arranged up and down to realize the refocusing of the cell, and the pins of the electrodes are connected to the output port of the power amplifier. When the flow channel is blocked, the pressure relief port 6 is used to clear the flow channel. The laser optical fiber 7 is connected to the LED light source to generate excitation light, and the detection optical fiber 8 is connected to the photomultiplier tube to detect the fluorescence signal. The blue-filled area 9 is an acoustic sorting electrode 9, and the pins of the electrode are connected to the output port of the power amplifier, which can push the target cells to the first sorting outlet 10 and the second sorting outlet 11. The first sorting outlet 10, the second sorting outlet 11 and the waste liquid outlet 12 are each connected to a needle hose to collect the sorted cells into a container for subsequent processing.

[0042] Figure 2 The flowchart of the microfluidic single cell sorting system based on acoustic focusing and sorting includes the following steps: S1, Microfluidic chip mounted on an inverted fluorescence microscope.

[0043] Specifically, the microfluidic chip is fixed on the moving loading platform of the inverted fluorescence microscope by a clamp; then a hollow steel needle with an outer diameter of 1.6 mm is inserted into the sheath liquid inlet 1 and the sample liquid inlet 2 of the microfluidic chip, and then one end of the hose is connected to the steel needle, and the other end is connected to the pressure pump of the matching flow meter, and the pressure pump is placed in the corresponding sheath liquid pool and sample liquid pool; continue to insert another hollow steel needle with an outer diameter of 1.6 mm into the first sorting outlet 10 and the second sorting outlet 11, and the waste liquid outlet 12, and one end of the hose is connected to the steel needle, and the other end is connected to the corresponding collection pool.

[0044] S2, turn on the power and set the system parameters.

[0045] Specifically, it is necessary to turn on the inverted fluorescence microscope, pressure pump, flowmeter, LED light source connected to the laser optical fiber 7, photomultiplier tube connected to the detection optical fiber 8, signal generator, power amplifier, DC power supply, data acquisition card and other power supplies, set the signal pulse waveform of the signal generator, including the pulse frequency, signal duration and delay trigger time; set the size of the pressure pump and flowmeter to control the injection speed of the sheath liquid and the sample liquid.

[0046] S3, the photomultiplier tube receives the fluorescence signal and outputs a corresponding level signal.

[0047] Specifically, the photomultiplier tube connected to the detection optical fiber 8 is used as a photon counting type fluorescence signal detector, which converts the optical signal into an electrical signal, so the intensity of the optical signal corresponds to the number of output level signals. The stronger the fluorescence signal, the more photons are output per unit time.

[0048] S4, the data acquisition card records the number of levels output by the photomultiplier tube.

[0049] Specifically, the counting port of the data acquisition card can continuously record the accumulated values ​​of the TTL level signal output by the photomultiplier tube for collection and processing by the host computer.

[0050] S5, the host computer calculates the photon increment and identifies the fluorescence signal.

[0051] Specifically, the host computer collects the accumulated number of photons at the counting port of the data acquisition card at a preset sampling time interval, subtracts the accumulated value at the previous moment, and obtains the accumulated number of photons at the sampling time interval; finally, the fluorescence recognition algorithm is used to identify whether there are stained cells flowing through the fluorescence detection area.

[0052] S6, the host computer outputs the sound wave sorting command to the data acquisition card.

[0053] Specifically, if the host computer detects stained cells, it immediately outputs a sorting command to the data acquisition card.

[0054] S7, the signal generator receives an external trigger command and outputs an electrical signal.

[0055] Specifically, after the signal generator receives the sorting pulse signal output by the data acquisition card, it immediately outputs a preset electrical signal to the power amplifier.

[0056] S8, the power amplifier amplifies the electrical signal.

[0057] Specifically, the power amplifier amplifies the electrical signal output by the signal generator, and the amplified signal directly acts on the acoustic wave sorting electrode 9 of the microfluidic chip.

[0058] S9, corresponding cells are sorted to the target outlet by the acoustic radiation force.

[0059] Specifically, when the stained cells flow through the acoustic wave sorting electrode 9, they are sorted to the corresponding target outlets due to the acoustic wave radiation force.

[0060] The system uses hydraulic focusing, acoustic focusing and acoustic sorting to achieve cell sorting and enrichment through fluorescence signal detection. The fluorescence detection module uses a laser optical fiber connected to an LED as an excitation light source, and a detection optical fiber connected to a photon-type photomultiplier tube for fluorescence signal detection, and proposes a fluorescence detection method for low data volume collection. The present invention reduces system cost, enhances integration, improves the signal-to-noise ratio of the signal, and contributes to the wide application of the system.

[0061] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A microfluidic single cell sorting system based on acoustic wave focusing and sorting, characterized in that: The system includes a microfluidic chip, an acoustic wave sorting module, and a fluorescence detection module; The microfluidic chip comprises a substrate, a hydraulic focusing structure for cell focusing, an acoustic wave focusing channel (5), and a sorting channel, wherein the hydraulic focusing structure, the acoustic wave focusing channel (5), and the sorting channel are connected in sequence; The acoustic wave sorting module comprises two acoustic wave focusing electrodes (4) and an acoustic wave sorting electrode (9) for cell sorting, wherein the two acoustic wave focusing electrodes (4) are connected to both sides of the acoustic wave focusing flow channel (5) on the substrate near the hydraulic focusing structure, and the acoustic wave sorting electrode (9) is connected to one side of the acoustic wave focusing flow channel (5) on the substrate and to the intersection of the acoustic wave focusing flow channel (5) and the sorting flow channel, and the number of the acoustic wave sorting electrodes (9) is the same as the number of the sorting flow channels; The fluorescence detection module comprises a laser optical fiber (7) connected to an LED light source, a detection optical fiber (8) connected to a photomultiplier tube, a data acquisition card for real-time recording the number of level signals output by the photomultiplier tube, a host computer for reading data from the data acquisition card, and an inverted fluorescence microscope for fixedly mounting the microfluidic chip. The laser optical fiber (7) and the detection optical fiber (8) are located on one side of the acoustic wave focusing channel (5), between the acoustic wave focusing electrode (4) and the acoustic wave sorting electrode (9).

2. The microfluidic single cell sorting system based on acoustic wave focusing and sorting according to claim 1, characterized in that: The acoustic wave sorting module also includes a signal generator for outputting an electrical signal, a power amplifier for amplifying the output signal of the signal generator, and a direct current power supply for providing a working voltage for the power amplifier.

3. The microfluidic single cell sorting system based on acoustic wave focusing and sorting according to claim 1, characterized in that: The hydraulic focusing structure comprises a sheath liquid inlet (1), a sample liquid inlet (2), a hydraulic focusing point (17), a sheath liquid delivery channel, and a hydraulic focusing flow channel (3); the sheath liquid inlet (1) is connected to the sheath liquid delivery channel, and the sheath liquid delivery channel is transformed from one inlet into a first sheath liquid delivery channel (13) and a second sheath liquid delivery channel (14) symmetrical with respect to the hydraulic focusing flow channel; the sample liquid inlet (2) is connected to the hydraulic focusing flow channel (3); the ends of the first sheath liquid delivery channel (13), the second sheath liquid delivery channel (14), and the hydraulic focusing flow channel (3) intersect and penetrate to form a hydraulic focusing point (17); the hydraulic focusing point (17) is connected to the acoustic wave focusing flow channel (5); and the ends of the acoustic wave focusing flow channel (5) are respectively connected to a waste liquid outlet (12) and a sorting flow channel.

4. The microfluidic single cell sorting system based on acoustic wave focusing and sorting according to claim 3, characterized in that: The acoustic wave sorting electrodes (9) are provided with two electrodes, which are respectively located on both sides of the acoustic wave focusing channel (5); the acoustic wave focusing channel (5) is connected to a first sorting outlet (10) and a second sorting outlet (11) respectively near the end through a first sorting channel (15) and a second sorting channel (16) of the same width for collecting target liquid; and the middle part near the end is connected to a waste liquid outlet (12).

5. The microfluidic single cell sorting system based on acoustic wave focusing and sorting according to claim 1, characterized in that: A pressure relief port (6) is provided between the acoustic wave focusing electrode (4) and the laser optical fiber (7) on the acoustic wave focusing channel (5), and the pressure relief port (6) is located on a side of the laser optical fiber and the detection optical fiber close to the hydraulic focusing structure.

6. The microfluidic single cell sorting system based on acoustic wave focusing and sorting according to claim 1, characterized in that: The sheath liquid inlet (1) and the sample liquid inlet (2) are both provided with a filtering microcolumn array.

7. The microfluidic single cell sorting system based on acoustic wave focusing and sorting according to claim 1, characterized in that: The acoustic wave focusing electrode (4) and the acoustic wave sorting electrode (9) are interdigitated electrodes.

8. The microfluidic single cell sorting system based on acoustic wave focusing and sorting according to claim 1, characterized in that: The substrate is assembled by bonding a lithium niobate substrate and polydimethylsiloxane by chemical bonding.

9. A microfluidic single cell sorting method based on acoustic wave focusing and sorting, characterized in that: Using the sorting system according to claim 2, comprising the following steps: Step S1, the microfluidic chip is mounted on an inverted fluorescence microscope; Step S2, turning on the power supply and setting the parameters of each electronic instrument; Step S3, the photomultiplier tube receives the fluorescence signal and outputs a corresponding level signal; Step S4, the data acquisition card records the number of levels output by the photomultiplier tube; Step S5, the host computer calculates the photon increment and identifies the fluorescence signal; Step S6, the host computer outputs the sound wave sorting command to the data acquisition card; Step S7, the signal generator receives an external trigger command and outputs an electrical signal; Step S8, the power amplifier amplifies the electrical signal; Step S9: The corresponding cells are sorted to the target outlet by the acoustic radiation force.

10. The microfluidic single cell sorting method based on acoustic wave focusing and sorting according to claim 9, characterized in that: The step S2 is to turn on the power supply, set the signal pulse waveform of the signal generator, including the pulse frequency, signal duration and delay trigger time, and adjust the injection speed of the sheath liquid and the sample liquid; In step S4, the counting port of the data acquisition card can continuously record the accumulated values ​​of the TTL level signal output by the photomultiplier tube for collection and processing by the host computer; In step S5, the host computer collects the accumulated number of photons at the counting port of the data acquisition card at a preset sampling time interval, subtracts the accumulated value at the previous moment, and obtains the accumulated number of photons at the sampling time interval; finally, the fluorescence recognition algorithm is used to identify whether there are stained cells flowing through the fluorescence detection area; In step S6, if the host computer detects stained cells, it immediately outputs a sorting command to the data acquisition card; In step S7, after the signal generator receives the sorting pulse signal output by the data acquisition card, it immediately outputs a preset electrical signal to the power amplifier; In step S8, the power amplifier amplifies the electrical signal output by the signal generator, and the amplified signal directly acts on the acoustic wave sorting electrode (9) of the microfluidic chip; In the step S9, when the stained cells flow through the acoustic wave sorting electrode (9), they are sorted to the corresponding target outlet under the action of the acoustic wave radiation force.

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